Methods on LDL particle isolation, characterization, and component fractionation for the development of novel specific oxidized LDL status markers for atherosclerotic disease risk assessment

The present study uses simple, innovative methods to isolate, characterize and fractionate LDL in its main components for the study of specific oxidations on them that characterize oxidized low-density lipoprotein (oxLDL) status, as it causatively relates to atherosclerosis-associated cardiovascular disease (CVD) risk assessment. These methods are: (a) A simple, relatively time-short, low cost protocol for LDL isolation, to avoid shortcomings of the currently employed ultracentrifugation and affinity chromatography methodologies. (b) LDL purity verification by apoB100 SDS-PAGE analysis and by LDL particle size determination; the latter and its serum concentration are determined in the present study by a simple method more clinically feasible as marker of CVD risk assessment than nuclear magnetic resonance. (c) A protocol for LDL fractionation, for the first time, into its main protein/lipid components (apoB100, phospholipids, triglycerides, free cholesterol, and cholesteryl esters), as well as into LDL carotenoid/tocopherol content. (d) Protocols for the measurement, for the first time, of indicative specific LDL component oxidative modifications (cholesteryl ester-OOH, triglyceride-OOH, free cholesterol-OOH, phospholipid-OOH, apoB100-MDA, and apoB100-DiTyr) out of the many (known/unknown/under development) that collectively define oxLDL status, which contrasts with the current non-specific oxLDL status evaluation methods. The indicative oxLDL status markers, selected in the present study on the basis of expressing early oxidative stress-induced oxidative effects on LDL, are studied for the first time on patients with end stage kidney disease on maintenance hemodialysis, selected as an indicative model for atherosclerosis associated diseases. Isolating LDL and fractionating its protein and main lipid components, as well as its antioxidant arsenal comprised of carotenoids and tocopherols, paves the way for future studies to investigate all possible oxidative modifications responsible for turning LDL to oxLDL in association to their possible escaping from LDL’s internal antioxidant defense. This can lead to studies to identify those oxidative modifications of oxLDL (after their artificial generation on LDL), which are recognized by macrophages and convert them to foam cells, known to be responsible for the formation of atherosclerotic plaques that lead to the various CVDs

The impact of kidney injury mediators on nitric oxide system on human tubular and mesenchymal kidney cells

The progression of renal fibrosis is characterized by loss of kidney function and deposition of extracellular matrix components. The mechanism implicated in the development of renal fibrosis involves cytokines and growth factors originating from endothelial, tubular epithelial and interstitial cells. Activated myofibloblasts derive from differentiated tubular epithelial cells trough a process called epithelial to mesenchymal transition. Various kidney injury mediators like cyclosporine‐A (CsA) are entering the luminal space of the tubules causing activation of profibrotic factors such as nitric oxide (NO) and endothelin‐1 (ET‐1). A cross talk exists between endothelin and NO systems in the regulation of vascular tone and inflammatory process. Aim of this study was to investigate the effect of cyclosporine‐A on the expression of Nitric Oxide and endothelin‐1 on cultured renal tubular cells. Human tubular epithelial cells (HK‐2) were cultured in the presence of CsA at various concentrations (0‐1,000 ng/ml). RT‐PCR was used to determine NO synthases (eNOS, iNOS) and endothelin receptors (ETR‐A, ETR‐B) and Western Blot analysis for the subsequent proteins. Similar experiments were also carried out using specific NO (L‐NAME) and endothelin receptor (BQ123, BQ 788) inhibitors. At therapeutic concentrations, CsA exerts a significant cytotoxic effect on tubular epithelial cells. A dose dependent activation of NO synthases eNOS and iNOS and endothelin receptors ET‐A and ETR‐B was observed, even at therapeutic concentrations of CsA. An interaction between NO and ET‐1 systems under the influence of CsA was also observed, since blockage of NO production was followed by down‐regulation of ET‐B while blocking of endothelin pathway with ET receptor antagonists, was followed by down‐regulation of eNOS expression.Η εξέλιξη της χρόνιας νεφρικής νόσου χαρακτηρίζεται από προοδευτική απώλεια της νεφρικής λειτουργίας και εναπόθεση εξωκυττάριας ύλης που οδηγεί σε γενικευμένη ίνωση του νεφρικού ιστού. Στους μηχανισμούς που ενοχοποιούνται για την εξέλιξη της βλάβης αυτής προς ίνωση συμμετέχουν κυτταροκίνες και αυξητικοί παράγοντες που προέρχονται από ενδοθηλιακά, επιθηλιακά σωληναριακά κύτταρα και κύτταρα του διάμεσου νεφρικού ιστού, ενώ συνυπάρχει και de novo ενεργοποίηση μυοϊνοβλαστών με τελικό αποτέλεσμα την αυξημένη εναπόθεση εξωκυττάριας ύλης. Τα επιθηλιακά σωληναριακά κύτταρα είναι πηγή προέλευσης των ενεργοποιημένων μυοϊνοβλαστών μέσω μιας διαδικασίας που ονομάζεται επιθηλιακή προς μεσεγχυματική μετάπτωση. Διάφοροι μεσολαβητές της νεφρικής βλάβης όπως είναι και η κυκλοσπορίνη ερχόμενοι σε επαφή με κύτταρα του εγγύς εσπειραμένου σωληναρίου οδηγούν σε ενεργοποίηση προϊνωτικών παραγόντων εκκρίνοντας εξωκυττάρια ύλη. Μεταξύ αυτών των μεσολαβητών της νεφρικής βλάβης, ιδιαίτερο ρόλο φαίνεται ότι διαδραματίζουν το νιτρικό οξείδιο και η ενδοθηλίνη, δύο παράγοντες που μετέχουν σε φυσιολογικές, αλλά και παθοφυσιολογικές κυτταρικές διαδικασίες. Τα συστήματα νιτρικού οξειδίου και ενδοθηλίνης αλληλεπιδρούν μεταξύ τους σε επίπεδο υποδοχέων στο κυτταρικό επίπεδο προκαλώντας αλλαγές του αγγειακού τόνου και ενεργοποίηση ή αναστολή προϊνωτικών σημάτων. Ο σκοπός της εργασίας ήταν η μελέτη της έκφρασης των συστημάτων νιτρικού οξειδίου και ενδοθηλίνης σε σωληναριακά νεφρικά κύτταρα κάτω από την επίδραση της κυκλοσπορίνης. Χρησιμοποιήθηκαν ανθρώπινες κυτταροκαλλιέργειες νεφρικών επιθηλιακών κυττάρων του εγγύς εσπειραμένου σωληναρίου (ΗΚ‐2) τα οποία επωάσθηκαν σε θεραπευτικές και τοξικές συγκεντρώσεις κυκλοσπορίνης (CsA) και ακολούθησε η ανίχνευση του νιτρικού οξειδίου (NO), της ενδοθηλιακής και επαγώγιμης συνθετάσης του ΝΟ (e‐NOS, i‐NOS) και της ενδοθηλίνης‐1 (ET‐1) με τους Α και Β υποδοχείς της (ΕΤ‐Α, ΕΤ‐Β). Το ΝΟ μετρήθηκε σύμφωνα με τη μέθοδο Griess, ενώ η συσσώρευση της ενδοθηλίνης και των Α και Β υποδοχέων της καθώς και οι συνθετάσες του ΝΟ ανιχνεύθηκαν τόσο σε επίπεδο μεταγράφου (m‐RNA) χρησιμοποιώντας RT‐PCR, όσο και σε επίπεδο πρωτεΐνης με Western Blot ανάλυση. Πειράματα πραγματοποιήθηκαν επίσης χρησιμοποιώντας ταυτόχρονα στο επωαστικό μέσο εκτός από κυκλοσπορίνη και ειδικούς αναστολείς του ΝΟ (L‐NAME) και των υποδοχέων της ενδοθηλίνης (BQ123, BQ 788), ενώ ακολούθησε η ανίχνευση των συνθετασών του ΝΟ και των υποδοχέων της ενδοθηλίνης αντίστοιχα. Από τα πειράματα που πραγματοποιήθηκαν προκύπτει ότι η κυκλοσπορίνη ασκεί τοξική δράση σε νεφρικά σωληναριακά κύτταρα και επάγει τη συσσώρευση της ΕΤ‐1, του ΝΟ, των συνθετασών του ΝΟ και των ΕΤ‐Α και ΕΤ‐Β υποδοχέων της ΕΤ‐1. Η επαγωγή του ΕΤ‐Α υποδοχέα είναι ανεξάρτητη από την παρουσία ή μη ΝΟ, σε αντίθεση με τον ΕΤ‐Β η επαγωγή του οποίου καταστέλλεται πλήρως όταν αναστέλλεται το σύστημα του νιτρικού οξειδίου (L‐NAME). Η επαγωγή της e‐NOS από την κυκλοσπορίνη είναι απόλυτα εξαρτώμενη από το σύστημα της ΕΤ‐1, σε αντίθεση με την i‐NOS η οποία επάγεται σε σημαντικό βαθμό ακόμη και όταν το σύστημα ενδοθηλίνης αδρανοποιείται πλήρως με ειδικούς αναστολείς (BQ123 και BQ788)

From basic anatomic configuration to maturation success

The arteriovenous fistula is the preferred vascular access for hemodialysis patients because of its low complication rate and lower costs, but it still has unacceptable failure rates. Krishnamoorthy et al. implicate the geometry of the fistula in the temporal and spatial variations occurring in two of the most important parameters of fistula maturation, blood flow and vessel diameter

Therapeutic Advances in Diabetic Kidney Disease

Although sodium glucose co-transporter type 2 (SGLT-2) inhibitors were initially introduced as glucose-lowering medications, it was later discovered that cardiorenal protection is the most important treatment effect of these agents. A triad of landmark trials consistently showed the benefits of SGLT-2 inhibitors on kidney and cardiovascular outcomes in patients with chronic kidney disease (CKD), irrespective of the presence or absence of Type 2 diabetes (T2D). Furthermore, finerenone is a novel, selective, nonsteroidal mineralocorticoid receptor antagonist (MRA) that safely and effectively improved cardiorenal outcomes in a large Phase 3 clinical trial program that included >13,000 patients with T2D and a wide spectrum of CKD. These two drug categories have shared and distinct mechanisms of action, generating the hypothesis that an overadditive cardiorenal benefit with their combined use may be biologically plausible. In this article, we describe the mechanism of action, and we provide an overview of the evidence for cardiorenal protection with SGLT-2 inhibitors and the nonsteroidal MRA finerenone in patients with CKD associated with T2D

Membranoproliferative glomerulonephritis in a patient with chronic brucellosis

Brucellosis is the most common zoonotic disease in Greece, with an endemic distribution and can affect any organ. Infiltration of the renal parenchyma causes acute and chronic interstitial nephritis with granulomas, whereas renal glomeruli are rarely affected. The disease has been sporadically reported, and it causes various histopathologic patterns. Herein, we describe the case of a 39-year-old stock breeder with a history of recurrent episodes of bacteremia caused by Brucella melitensis over a period of 3 years. Two months after the last episode of bacteremia, he presented with mild renal insufficiency, nephrotic range proteinuria, and microscopic hematuria. A renal biopsy revealed membranoproliferative glomerulonephritis with a pattern of focal-segmental nodular sclerosis and moderate tubulointerstitial fibrosis. The patient received antimicrobial and corticosteroid therapy with partial remission of the nephrotic syndrome

Neutrophil Gelatinase-Associated Lipocalin as a Biomarker of Acute Kidney Injury in Patients with Morbid Obesity Who Underwent Bariatric Surgery

Introduction: Neutrophil gelatinase-associated lipocalin (NGAL) has been identified as a biomarker of acute kidney injury (AKI) that could contribute to early diagnosis and effective management of AKI. The purpose of this study was to evaluate NGAL as a predictive marker of AKI in patients with clinically severe obesity (BMI >50) who underwent biliopancreatic diversion surgery. Patients and Methods: We prospectively studied 23 patients with clinically severe obesity who underwent biliopancreatic bypass surgery. NGAL was measured using chemiluminescent microparticle immunoassay in three urine samples collected from each patient before surgery (first sample), 2-6 h after surgery (second sample) and on the first postoperative day (third sample). Results: Renal function was evaluated using serum creatinine values, which were 0.91 ± 0.26 mg/dl before surgery, 0.87 ± 0.31 mg/dl immediately after surgery and 0.92 ± 0.62 mg/dl on the fifth postoperative day. During the immediate postoperative period, AKI was observed in 2 patients, one of whom required renal replacement therapy with hemodialysis. In both patients, urine NGAL increased within the first postoperative hours before the values of serum creatinine increased. Conclusion: Urine NGAL in patients with clinically severe obesity, which was surgically treated, might be a potential biomarker of early AKI detection. Further research is required in order to confirm these results observed in a small number of patients who developed postoperative AKI and make recommendations for predictive NGAL values in patients who underwent bariatric surgery

High levels of synovial fluid osteoprotegenin (OPG) and increased serum ratio of receptor activator of nuclear factor-kappa B ligand (RANKL) to OPG correlate with disease severity in patients with primary knee osteoarthritis

Objective: Evaluation of serum and synovial fluid OPG and sRANKL in 37 patients with primary knee osteoarthritis. Design and method: OPG and sRANKL were measured using ELISA. Results: OPG, sRANKL and sRANKL/OPG were increased in osteoarthritis patients’ serum. Synovial OPG was higher than serum OPG, while sRANKL/OPG was higher in the serum; both correlated with disease severity. Discussion: RANKL/OPG pathway is implicated in the pathogenesis of knee osteoarthritis being a suitable target for therapeutic intervention. (c) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved

Increased Prevalence and Severity of Coronary Artery Calcification in Patients with Chronic Kidney Disease Stage III and IV

Background: Cardiovascular disease (CVD) is the main cause of mortality in patients with chronic kidney disease (CKD). The pathophysiology of coronary artery disease in CKD is multifactorial including, in addition to traditional risk factors (hypertension, hyperlipidemia, diabetes mellitus), parameters related to uremia. Methods: The study consisted of measuring coronary artery calcification (CAC) score in patients with CKD stage III and IV without history of CVD and in a group of controls with normal renal function matched for age, gender and risk factors using multi-detector computed tomography. Results: The study included 49 patients and 49 controls. CAC was present in 79.6% in the CKD group versus 59.2% in the control group (p = 0.028). The median CAC score value in CKD patients was 139 (interquartile range (IQR): 23–321) versus 61 (IQR: 6–205) in controls (p = 0.007). CAC was associated with traditional risk factors such as older age, hypertension and baseline cardiovascular risk score, while CKD patients with severe calcification had marginally lower estimated glomerular filtration rate and increased levels of parathormone. Conclusions: CAC is more frequent and severe in patients with CKD stage III and IV compared to matched controls with normal renal function, even though kidney disease-related parameters are not directly correlated with intensity of calcification

Time Series Network Data Enabling Distributed Intelligence—A Holistic IoT Security Platform Solution

The Internet of Things (IoT) encompasses multiple fast-emerging technologies controlling and connecting millions of new devices every day in several application domains. The increased number of interconnected IoT devices, their limited computational power, and the evolving sophistication of cyber security threats, results in increased security challenges for the IoT ecosystem. The diversity of IoT devices, and the variety of QoS requirements among several domains of IoT application, impose considerable challenges in designing and implementing a robust IoT security solution. The aim of this paper is to present an efficient, robust, and easy-to-use system, for IoT cyber security operators. Following a by-design security approach, the proposed system is a platform comprising four distinct yet cooperating components; a distributed AI-enhanced detection of potential threats and anomalies mechanisms, an AI-based generation of effective mitigation strategies according to the severity of detected threats, a system for the verification of SDN routing decisions along with network- and resource-related policies, and a comprehensive and intuitive security status visualization and analysis. The distributed anomaly detection scheme implementing multiple AI-powered agents is deployed across the IoT network nodes aiming to efficiently monitor the entire network infrastructure. Network traffic data are fed to the AI agents, which process consecutive traffic samples from the network in a time series analysis manner, where consecutive time windows framing the traffic of the surrounding nodes are processed by a graph neural network algorithm. Any detected anomalies are handled by a mitigation engine employing a distributed neural network algorithm, which exploits the recorded anomalous events and deploys appropriate responses for optimal threat mitigation. The implemented platform also includes the hypothesis testing module, and a multi-objective optimization tool for the quick verification of routing decisions. The system incorporates visualization and analytics functionality and a customizable user interface